Directed switch-mediated DNA recombination

ABSTRACT

Switch regions derived from an immunoglobulin (Ig) gene are used to direct recombination between a targeting construct containing a promoter, a switch region (S1), and 2) a target locus minimally containing a promoter, a switch region (S2), and a target sequence.

This application is a continuation of application Ser. No. 08/619,109,filed Mar. 20, 1996, U.S. Pat. No. 5,714,352.

FIELD OF THE INVENTION

This invention relates generally to methods and compositions for use inrecombinant DNA technology, particularly in methods for manipulation ofDNA sequences encoding antibodies, proteins, or portions thereof.

BACKGROUND OF THE INVENTION

The basic immunoglobulin (Ig) structural unit in vertebrate systems iscomposed of two identical "light" polypeptide chains (approximately 23kDa), and two identical "heavy" chains (approximately 53 to 70 kDa). Thefour chains are joined by disulfide bonds in a "Y" configuration, andthe "tail" portions of the two heavy chains are bound by covalentdisulfide linkages when the immunoglobulins are generated either by Bcell hybridomas or other cell types.

A schematic of the general antibody structure is shown in FIG. 1. Thelight and heavy chains are each composed of a variable region at theN-terminal end, and a constant region at the C-terminal end. In thelight chain, the variable region (termed "V_(L) J_(L) ") is composed ofa variable (V_(L)) region connected through the joining (J_(L)) regionto the constant region (C_(L)). In the heavy chain, the variable region(V_(H) D_(H) J_(H)) is composed of a variable (V_(H)) region linkedthrough a combination of the diversity (D_(H)) region and the joining(J_(H)) region to the constant region (C_(H)). The V_(L) J_(L) and V_(H)D_(H) J_(H) regions of the light and heavy chains, respectively, areassociated at the tips of the Y to form the antibody's antigen bindingportion and determine antigen binding specificity.

The (C_(H)) region defines the antibody's isotype, i.e., its class orsubclass. Antibodies of different isotypes differ significantly in theireffector functions, such as the ability to activate complement, bind tospecific receptors (e.g., Fc receptors) present on a wide variety ofcell types, cross mucosal and placental barriers, and form polymers ofthe basic four-chain IgG molecule.

Antibodies are categorized into "classes" according to the C_(H) typeutilized in the immunoglobulin molecule (IgM, IgG, IgD, IgE, or IgA).There are at least five types of C_(H) genes (Cμ, Cγ, Cδ, Cε, and Cα),and some species (including humans) have multiple C_(H) subtypes (e.g.,Cγ₁, Cγ₂, Cγ₃, and Cγ₄ in humans). There are a total of nine C_(H) genesin the haploid genome of humans, eight in mouse and rat, and severalfewer in many other species. In contrast, there are normally only twotypes of light chain constant regions (C_(L)) , kappa (κ) and lambda(λ), and only one of these constant regions is present in a single lightchain protein (i.e., there is only one possible light chain constantregion for every V_(L) J_(L) produced). Each heavy chain class can beassociated with either of the light chain classes (e.g., a C_(H) γregion can be present in the same antibody as either a κ or λ lightchain), although the constant regions of the heavy and light chainswithin a particular class do not vary with antigen specificity (e.g., anIgG antibody always has a Cγ heavy chain constant region regardless ofthe antibody's antigen specificity).

Each of the V, D, J, and C regions of the heavy and light chains areencoded by distinct genomic sequences. Antibody diversity is generatedby recombination between the different V_(H), D_(H), and J_(H) genesegments in the heavy chain, and V_(L) and J_(L) gene segments in thelight chain. The recombination of the different V_(H), D_(H), and J_(H)genes is accomplished by DNA recombination during B celldifferentiation. Briefly, the heavy chain sequence recombines first togenerate a D_(H) J_(H) complex, and then a second recombinatorial eventproduces a V_(H) D_(H) J_(H) complex. A functional heavy chain isproduced upon transcription followed by splicing of the RNA transcript.Production of a functional heavy chain triggers recombination in thelight chain sequences to produce a rearranged V_(L) J_(L) region whichin turn forms a functional V_(L) J_(L) C_(L) region, i.e., thefunctional light chain.

During the course of B cell differentiation, progeny of a single B cellcan switch the expressed immunoglobulin isotype from IgM to IgG or otherclasses of immunoglobulin without changing the antigen specificitydetermined by the variable region. This phenomenon, known asimmunoglobulin class-switching, is accompanied by DNA rearrangement thattakes place between switch (S) regions located 5' to each C_(H) gene(except for Cγ) (reviewed in Honjo (1983) Annu. Rev. Immunol. 1:499-528,and Shimizu & Honjo (1984) Cell 36:801-803). S--S recombination bringsthe V_(H) D_(H) J_(H) exon to the proximity of the C_(H) gene to beexpressed by deletion of intervening C_(H) genes located on the samechromosome. The class-switching mechanism is directed by cytokines(Mills et al. (1995) J. Immunol. 155:3021-3036). Switch regions vary insize from 1 kb (Sε) to 10 kb (Sγ₁), and are composed of tandem repeatsthat vary both in length and sequence (Gritzmacher (1989) Crit. Rev.Immunol. 9:173-200). Several switch regions have been characterizedincluding the murine Sμ, Sε, Sα, Sγ3, Sγ1, Sγ2b and Sγ2a switch regionsand the human Sμ switch region (Mills et al. (1995) supra; Nikaido etal. (1981) Nature 292:845-8; Marcu et al. (1982) Nature 298:87-89;Takahashi et al. (1982) Cell 29:671-9; Mills et al. (1990) Nucleic AcidsRes. 18:7305-16; Nikaido et al. (1982) J. Biol. Chem. 257:7322-29;Stanton et al. (1982) Nucleic Acids Res. 10:5993-6006; Gritzmacher(1989) supra; Davis et al. (1980) Science 209:1360; Obata et al. (1981)Proc. Natl. Acad. Sci. U.S.A. 78:2437-41; Kataoka et al. (1981) Cell23:357; Mowatt et al. (1986) J. Immunol. 136:2674-83; Szurek et al.(1985) J. Immunol. 135:620-6; and Wu et al. (1984) EMBO J. 3:2033-40).

Observations that a single B cell can express more than one isotypesimultaneously on its surface is not explained by the class-switchingmechanism since S--S recombination is limited to intrachromosomalrecombination and results in deletion of the exchanged C_(H) gene. Asecond mechanism, called trans-splicing, has been described in which twotranscripts generated from different chromosomes are joined to form asingle continuous transcript (Shimizu et al. (1991) J. Exp. Med.173:1385-1393). Transgenic mice carrying a rearranged expressible V_(H)D_(H) J_(H) heavy chain μ gene integrated outside the mouse IgH locuswere found to produce mRNA having the V_(H) D_(H) J_(H) region of thetransgene correctly spliced to the endogenous C_(H) region. As with S--Srecombination, the frequency of trans-splicing is low, and the factorsregulating both mechanisms are not well understood.

The value and potential of antibodies as diagnostic and therapeuticreagents has been long-recognized in the art. Unfortunately, the fieldhas been hampered by the slow, tedious processes required to producelarge quantities of an antibody of a desired specificity. The classicalcell fusion techniques allowed for efficient production of monoclonalantibodies by fusing the B cell producing the antibody with animmortalized cell line. The resulting cell line is called a hybridomacell line. However, most of these monoclonal antibodies are produced inmurine systems and are recognized as "foreign" proteins by the humanimmune system. Thus the patient's immune system elicits a responseagainst the antibodies, which results in antibody neutralization andclearance, and/or potentially serious side-effects associated with theanti-antibody immune response.

One approach to this problem has been to develop human or "humanized"monoclonal antibodies, which are not as easily "recognized" as foreignepitopes, and avoid an anti-antibody immune response in the patient.Applications of human B cell hybridoma-produced monoclonal antibodieshave promising potential in the treatment of cancer, microbial, andviral infections, B cell immunodeficiencies associated with abnormallylow antibody production, autoimmune diseases, inflammation, transplantrejection and other disorders of the immune system, and other diseases.However, several obstacles remain in the development of such humanmonoclonal antibodies. For example, many human tumor antigens may not beimmunogenic in humans and thus it may be difficult to isolate human Bcells producing antibodies against human antigens.

Attempts to address the problems associated with antibodies for humantherapeutics have used recombinant DNA techniques. Most of these effortshave focused on the production of chimeric antibodies having a humanC_(H) region and non-human (e.g., murine) antigen combining (variable)regions. These chimeric antibodies are generally produced by cloning thedesired antibody variable region and/or constant region, combining thecloned sequences into a single construct encoding all or a portion of afunctional chimeric antibody having the desired variable and constantregions, introducing the construct into a cell capable of expressingantibodies, and selecting cells that stably express the chimericantibody. Alternatively, the chimeric antibody is produced by cloningthe desired variable region or constant region, introducing theconstruct into an antibody-producing cell, and selecting for chimericantibody-producing cells that result from homologous recombinationbetween the desired variable region and the endogenous variable region,or the desired constant region and the endogenous constant region.Examples of techniques which rely upon recombinant DNA techniques suchas those described above to produce chimeric antibodies are described inPCT Publication No. WO 86/01533 (Neuberger et al.), and in U.S. Pat.Nos. 4,816,567 (Cabilly et al.) and 5,202,238 (Fell et al.). Thesemethods require transferring DNA from one cell to another, thus removingit from its natural locus, and thus require careful in vitromanipulation of the DNA to ensure that the final antibody-encodingconstruct is functional (e.g., is capable of transcription andtranslation of the desired gene product).

There is a clear need in the field for a method for producing a desiredprotein or antibody which does not require multiple cloning steps, inmore efficient than conventional homologous recombination, and can becarried out in hybridoma cells.

SUMMARY OF THE INVENTION

The present invention features a method of replacing one DNA sequencewith another using switch (S) regions derived from an immunoglobulin(Ig) gene. The method of the invention allows any two pieces of DNA tobe "switched" or a piece of exogenous DNA to be inserted into a sitecontaining a natural or artificial S region. Thus the method of theinvention allows directed recombination to occur and eliminates manycloning steps required by current recombinant DNA methods.

In the method of the invention, directed recombination is brought aboutbetween a targeting construct and a target locus. The nucleic acidtargeting construct is composed minimally of a switch region and apromoter operably linked to and 5' of the switch region. Additionally,depending on the desired recombinatorial product, the targetingconstruct can also contain a modifying sequence operably linked to and3' of the switch region, and other DNA sequences between the promoterand switch regions, e.g., 5' of the switch region and 3' of the promoterregion. Of particular interest is the use of a targeting construct withan Ig heavy chain to facilitate isotype switching, e.g., replacement ofan endogenous constant region (C_(H)) in an antibody heavy chain gene(target sequence) with a C_(H) of a different subtype, isotype, orspecies of origin (modifying sequence). For example, exogenous DNAencoding the constant or variable region of an antibody light or heavychain can be switched with the constant or variable region of anendogenous sequence to create a sequence which encodes an antibody witha different constant or variable region. In a broader sense, the methodof the invention is widely applicable to manipulate DNA sequences forproduction of a desired protein or protein component, including theproduction of chimeric antibodies having a desired variable regionlinked to a non-antibody polypeptide (e.g., a detectable polypeptidelabel, or a polypeptide having a desired activity).

In one aspect, the invention features a method for directedswitch-mediated recombination by a) introducing a targeting constructinto a cell having a target locus, the target locus being minimallycomposed of a promoter, a switch region, and a target sequence, whereinthe targeting construct is minimally composed of a promoter and a switchregion, and can contain additional modifying sequences, b) culturing thecell to allow transcription of the target locus and the targetingconstruct, thereby promoting recombination of the switch regions of thetarget locus and the targeting construct, and c) selecting a cellcontaining the desired recombined DNA product sequence, minimallycomposed of a switch region (composed of DNA sequences from one or boththe target locus switch region and targeting construct switch region).

In a specific embodiment of the invention, the targeting construct (P₁-S₁) is composed of a promoter (P₁) and switch region (S₁) and thetarget locus (P₂ -S₂ -T) is composed of a promoter (P₂), a naturallyoccurring or artificially inserted switch region (S₂), and a targetsequence (T). Directed S--S recombination between the S--S regionsresults in a DNA sequence having the P₁ promoter of the targetingconstruct, a switch region containing DNA sequences from one or both S₁and S₂ regions, and the T sequence (P₁ -S₁ /S₂ -T). In this embodiment,the target sequence is removed from the control of the target locuspromoter and placed under control of the desired P₁ promoter. Cellscontaining the desired DNA sequence are selected by methods known in theart, including Southern blot analysis or PCR.

In another embodiment, the targeting construct (P₁ -S₁ -M) is composedof a promoter (P₁), a switch region (S₁), and a modifying sequence (M),and the target locus (P₂ -S₂ -T) is composed of a promoter (P₂), anaturally occurring or artificially inserted switch region (S₂), and atarget sequence (T). Directed S--S recombination between the S--Sregions results in two possible recombinatorial product sequences, onehaving the P₁ promoter of the targeting construct, a switch regioncontaining DNA sequences from one or both S₁ and S₂ regions, and the Tsequence (P₁ -S₁ /S₂ -T), and a second sequence having a P₂ promoter, aswitch region containing DNA sequences from one or both S₁ and S₂regions, and the M sequence (P₁ -S₁ /S₂ -M). In this embodiment, cellsexpressing the M sequence are selected by methods known in the art,including Southern or Northern blot analysis.

In a third embodiment, the targeting construct (P₁ -Z-S₁) is composed ofa promoter (P₁), DNA sequences 5' to the switch region (Z), and theswitch region (S₁). The target locus (P₂ -S₂ -T) is composed of apromoter (P₂), a naturally occurring or artificially inserted switchregion (S₂), and a target sequence (T). Directed S--S recombinationbetween the switch regions results in a DNA sequence having the P₁promoter of the targeting construct, the Z DNA sequences, a switchregion containing DNA sequences from one or both switch regions, and theT sequence (P₁ -Z-S₁ /S₂ -T).

The target locus is a DNA sequence having a switch region, and may be anative, naturally-occurring sequence (e.g., an Ig locus of anantibody-producing cell), a rearranged Ig locus, or a recombinantlyproduced DNA sequence artificially inserted at a desired site. Thetarget locus can be either an extrachromosomal element or a stablyintegrated chromosomal element. Preferably, the target locus encodes anantibody heavy chain gene. The targeting construct is either anextrachromosomal element or a stably integrated chromosomal element.Where the target locus is an antibody heavy chain gene, the modifyingsequence of the targeting construct preferably encodes a different ormodified heavy chain constant region or a non-antibody sequence ofinterest (e.g., a detectable polypeptide label, an enzyme, a toxin, or agrowth factor).

The invention provides a method of modifying a DNA sequence by directedS--S recombination. The invention allows DNA recombination to bedirected to any site which contains a naturally-occurring switch regionor synthetic switch region, including a site into which an S region hasbeen artificially inserted.

The invention provides a method to replace or modify a first DNAsequence (a target sequence) with a second DNA sequence (a modifyingsequence) without the need for isolating the nucleotide sequencecontaining the target sequence, excising the target sequence, andligating the modifying sequence in place of the target sequence. Theinvention also provides a method to replace portions of apolypeptide-encoding sequences with a heterologous amino acid sequence,where the polypeptide is composed of two distinct components (e.g., anN-terminal component and a C-terminal component) that, for example,confer distinct functional or structural characteristics upon thepolypeptide (e.g., ligand binding or cell-binding). For example, theinvention allows for the substitution of either the N-terminal portionwith a different, heterologous amino acid-encoding sequence, or theC-terminal portion with a different, heterologous amino acid-encodingsequence.

Directed switch-mediated recombination allows recombination to occur ata specific, pre-selected region with an increased efficiency relative tothe naturally-occurring mechanism which is limited to the immunoglobulinheavy chain. The method of the invention removes switch-mediatedrecombination from the limitations of its normal regulatory environment,allowing recombination to be controlled as needed with, for example, theuse of constitutive or inducible promoters.

The ability to accomplish directed in vitro S-mediated recombinationavoids tedious, time-consuming manipulation of DNA using conventionalrecombinant DNA techniques while providing a highly efficient method ofinserting a DNA sequence. For example, the method allows the detectablelabel portion of fusion proteins (e.g., β-galactosidase) to be readilyexchanged for a different amino acid sequence (e.g., alkalinephosphatase).

In a specific application of the method of the invention, directed S--Srecombination is used to replace the constant region of an antibodyheavy chain gene with a different or modified constant region withoutthe need for extensive manipulation of the antibody heavy chain gene.Additionally, the method of the invention allows the antibody gene to bemaintained in its native locus.

These and other objects, advantages and features of the presentinvention will become apparent to those persons skilled in the art uponreading the details of the compositions, composition components, methodsand method steps of the invention as set forth below.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic showing the basic immunoglobulin structure.

FIG. 2A is a schematic showing the basic components of a target locusconsisting of a promoter (P₂), switch region (S₂), and a target sequence(T).

FIG. 2B is a schematic showing the basic components of a target locusconsisting of a promoter (P₂), DNA sequences positioned 3' of thepromoter and 5' of the switch region (Y), switch region (S₂), and atarget sequence (T).

FIG. 3A is a schematic showing the basic components of a targetingconstruct consisting of a promoter (P₁) and switch region (S₁).

FIG. 3B is a schematic showing the basic components of a targetingconstruct consisting of a promoter (P₁), switch region (S₁), andmodifying sequences.

FIG. 3C is a schematic showing the basic components of a targetingconstruct consisting of a promoter (P₁), DNA sequences positioned 3' ofthe promoter and 5' of the switch region (Z), and switch region (S₁).

FIG. 3D is a schematic showing the basic components of a targetingconstruct consisting of a promoter (P₁), DNA sequences positioned 3' ofthe promoter and 5' of the switch region (Z), switch region (S₁), andmodifying sequences, which may include additional components such as aselectable marker gene and/or an amplification gene.

FIG. 4A is a schematic illustrating switch-mediated recombinationbetween targeting construct P₁ -S₁ and target locus P₂ -S₂ -T.

FIG. 4B is a schematic illustrating switch-mediated recombinationbetween targeting construct P₁ -S₁ -M and target locus P₂ -S₂ -T.

FIG. 4C is a schematic illustrating switch-mediated recombinationbetween targeting construct P₁ -Z-S₁ and target locus P₂ -S₂ -T.

FIG. 5 is a schematic of a directed targeting construct of the invention(pTSW-1.4) having the entire 23 Kb human γ2locus (5' control elements, Iexon, switch regions, coding sequences, membrane and secretory exons,polyA), mouse 3' enhancer sequence, CMV promoter/enhancer cassette, andSV2 hygromycin selectable marker.

FIG. 6 is a schematic of a targeting construct of the invention(pTSW-1.9) with the elements as described in the legend to FIG. 5, withthe CMV promoter/enhancer cassette in the opposite orientation to thatof pTSW-1.4.

FIG. 7 is a schematic of a targeting construct of the invention (pTSW-2)having a 12 kb BamHI fragment cloned from the 23 kb human γ2 germlineclone (including switch regions and the human γ2 open reading frame; theI exon and 5' control elements are not included), CMV promoter/enhancercassette providing splice donor site, and SV2 hygromycin selectablemarker; the mouse 3' enhancer is not included.

FIG. 8 is a schematic of a targeting construct of the invention(pTSW-3.1) having 10 kb of cloned HindIII-EcoRI mouse γ1 genomic switchfragment (5' control elements, I exon, and mouse γ1 switch sequences),CMV promoter cassette (SFFV promoter cassette in the pTSW-3.2 series),genomic clone of human γ2 open reading frame and splice acceptor, SV2hygromycin selectable marker, and optionally the mouse 3' enhancersequence.

FIG. 9 is a schematic of a targeting construct of the invention(pTSW-3.1BglII) having 7.9 kb BglII-EcoRI mouse γ1 genomic switchfragment (I exon and mouse γ1 switch sequences; 5' control elements notincluded), CMV promoter cassette (SFFV promoter cassette in the pTSW-3.2series), genomic clone of human γ2 open reading frame and spliceacceptor, SV2 hygromycin selectable marker, and optionally the mouse 3'enhancer sequence.

DETAILED DESCRIPTION

Before the methods and compositions of the present invention aredescribed and disclosed it is to be understood that this invention isnot limited to the particular methods and compositions described as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting since the scope of the presentinvention will be limited only by the appended claims.

It must be noted that as used in this specification and the appendedclaims, the singular forms "a", "an" and "the" include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to "a DNA sequence" includes a plurality of DNA sequences anddifferent types of DNA sequences.

Unless defined otherwise all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any materials ormethods similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference for the purpose of describing anddisclosing the particular information for which the publication wascited in connection with. The publications discussed above are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventor is not entitled to antedate such disclosure by virtue of priorinvention.

Definitions

The term "artificial" as used with "artificial construct" or "artificialswitch region" and the like, refers to an isolated natural ornon-naturally occurring material e.g., a nucleotide sequencemanufactured by human intervention e.g., fusing natural sequencestogether or chemically synthesizing natural sequences in isolation.

The term "switch region" means a nucleotide sequence composed of tandemrepeat sequences that occur in nature 5' to the immunoglobulin heavychain constant region and function in intrachromosomal class-switching,i.e., recombination of DNA sequences encoding specific portions ofimmunoglobulin heavy chain constant regions. Examples of specific switchregion sequences are disclosed in Mills et al. (1995) J. Immunol.155:3021-3036, herein specifically incorporated by reference. "Switchregion" includes both full-length switch sequences of nativeimmunoglobulin sequences, as well as recombinant and syntheticnucleotide sequences that are modified (e.g., contain nucleotidesubstitutions, additions, mutations, and/or other modifications)relative to a native immunoglobulin switch region, with the proviso thatthe switch region retains its function in facilitating recombinationwhen transcribed.

The term "switch-mediated recombination" or "directed S--Srecombination" are used interchangeably to mean interchromosomal,intrachromosomal, or extrachromosomal DNA recombination facilitated by aswitch region. For example, S--S recombination results from interactionof 1) a first switch region positioned 3' to a promoter (targetingconstruct) and 2) a second switch region positioned 3' to a promoter and5' to a DNA sequence (target locus). Following activation oftranscription of the first and second switch regions, recombinationoccurs between the switch regions resulting in an alteration of thetarget locus DNA sequence. The directed S--S recombination of theinvention results in interaction between DNA sequences on two differentchromosomes, on the same chromosome, between a chromosome and anextrachromosomal element, or between two extrachromosomal elements.

The term "targeting construct" means a nucleic acid construct which isintroduced into a cell to cause directed S--S recombination at a naturalor artificial switch region. A targeting construct minimallycomprises: 1) a switch region and 2) a promoter operably linked to and5' of the switch region. Optionally, the targeting construct furthercomprises 3) a modifying sequence operably linked to and 3' of theswitch region. The targeting construct may also comprise 4) one or moreDNA sequences between the switch region and promoter. Depending on theactual targeting construct used, the resulting mRNA will encode theswitch region, or the switch region and the modifying sequence, or theswitch region and DNA sequences between the switch region and/or amodifying sequence.

The term "target locus" means a nucleic acid sequence minimallycomprises 1) a switch region, 2) a target sequence adjacent and 3' ofthe switch region, and 3) a promoter operably positioned in the targetlocus to provide transcription of the switch region and target sequenceas one or more translatable mRNA(s). The target locus can furthercontain an additional DNA sequence positioned adjacent and 5' of theswitch region; in such constructs, the promoter provides transcriptionof the additional DNA sequence, the switch region, and the targetsequence as one or more translatable mRNA(s). "Target loci" can beeither naturally occurring (e.g., an immunoglobulin gene composed of arearranged VDJ region positioned 5' of a switch region and C_(H) gene)or recombinantly or synthetically produced, and can be eitherchromosomal or extrachromosomally located. An exemplary target sequencecomprises a promoter sequence operatively positioned 5' to a switchregion operatively positioned 5' to a coding sequence which ispreferably a sequence encoding a constant region of a human antibody.

The terms "target sequence" means the nucleic acid sequence adjacent toa switch region where directed S--S recombination takes place. In oneembodiment of the method of the invention, a target sequence is replacedby the modifying sequence after switch-mediated recombination. "Targetsequences" can be naturally occurring sequences endogenous to achromosomal sequence or recombinant sequences (i.e., a sequence producedusing recombinant genetic manipulation) present as an extrachromosomalelement (e.g., a vector) or as a stably integrated element within achromosomal sequence. Target sequences are adjacent to a switch regionwhich may be a naturally occurring switch region or may be a switchregion inserted 5' to a desired target sequence by recombinant DNAtechnology. Exemplary target sequences are different from the modifyingsequence and include sequences encoding an immunoglobulin heavy chainconstant region of a particular isotype, subtype, and/or origin.

The term "immunoglobulin (Ig) locus" means a nucleotide sequence thatencodes all or a portion of the constant region and/or variable regionof an antibody molecule, including all or portions of the regulatorysequences that control expression of an antibody molecule from the locusor its processes. Heavy chain genes in Ig loci include but are notlimited to all or a portion of the V_(H), D_(H), J_(H), and constantregions, as well as the switch regions, intronic sequences, and flankingsequences associated with or adjacent the heavy chain gene. Ig loci forlight chains include but are not limited to the V_(L), J_(L), andconstant regions of both the kappa and lambda alleles, intronicsequences, and flanking sequences associated with or adjacent the lightchain gene.

The term "modified target locus" means a nucleic acid sequence modifiedby switch-mediated DNA recombination so that the modified targetsequence is minimally composed of a switch region composed of switchsequences derived from the unmodified target locus switch region, orfrom both the unmodified target locus and the targeting construct. Inone embodiment of the invention, the modified target locus is alsocomposed of the promoter of the unmodified target sequence, the firstDNA sequence of the unmodified target sequence (when present in theoriginal target locus), and the modifying sequence of the targetingconstruct. Activation of transcription by the promoter results intranscription of the first DNA sequence, the switch region, and themodifying sequence in one or more translatable mRNA(s).

The term "promoter" means a nucleotide sequence that, when operablylinked to a DNA sequence of interest, promotes transcription of that DNAsequence.

The term "detectable polypeptide label" means a amino acid sequencethat, when covalently bound to another amino acid sequence, provides aheterologous sequence that can be readily detected. For example, thepolypeptide can be detected by binding of a polypeptide-specificantibody, by virtue of an enzymatic activity of the polypeptide, or byreaction of the polypeptide with a chemical reagent. Exemplarydetectable polypeptide labels include β-galactosidase, alkalinephosphatase, horseradish peroxidase, enzymatically active portions ofthese enzymes, or any amino acid sequence that is immunodetectable andheterologous to the amino acid sequence with which it is associated.

Directed S--S Recombination (General)

The method of directed switch region-mediated recombination uses switchregions (e.g., those isolated and derived from an immunoglobulin locus)to facilitate recombination at a specific nucleic acid sequence. Thenucleic acid sequence to which S--S recombination is directed containsan S region and is termed a "target locus," while the introduced nucleicacid sequence containing a S region sequence is termed a "targetingconstruct." Transcription of each S region allows S--S recombination tooccur between the two preselected DNA regions. The presence of aselected promoter provides constitutive or inducible transcription,thereby enhancing the frequency of S--S recombination occurrence.

The basic components of an exemplary target locus suitable for use inthe invention are illustrated in FIG. 2A. The minimal components of thetarget locus are (from 5' to 3'): 1) a promoter (P₂, where the arrowindicates the direction of transcription), 2) a switch region (S₂), and3) a target sequence (T). Alternatively, the target locus can furthercontain an additional DNA sequence positioned 3' of the promoter and 5'of the switch region (Y) (FIG. 2B). Regardless of its composition, thetarget locus components are positioned so that the promoter activatestranscription of the 5' DNA sequence (optional), switch region, andtarget sequence (optional). The target locus can either be anendogenous, naturally-occurring chromosomal sequence (e.g., an Ig heavychain locus where the 5' DNA sequence is a V_(H) D_(H) J_(H) gene andthe target sequence is a C_(H) gene) or an artificially constructedsequence (i.e., a recombinantly produced sequence or a synthesizedsequence) which is present as either an extrachromosomal element (e.g.,a vector or plasmid) or as a stable chromosomal integrant.

The basic components of an exemplary targeting construct for use in theinvention are illustrated in FIGS. 3A-3D. The minimal components of thetargeting construct are (from 5' to 3'): 1) a promoter (P₁, where thearrow indicates the direction of transcription) and 2) a switch region(S) (FIG. 3A). The targeting construct can additionally contain 3) amodifying sequence 3' to S (FIG. 3B), and/or 4) one or more DNAsequences 5' to S (FIG. 3C). Additionally, the targeting construct caninclude a selectable marker (FIG. 3D). The targeting constructcomponents are positioned so that the promoter activates transcriptionof the switch regions and modifying sequence. The targeting construct isnormally a recombinantly or synthetically produced nucleic acidsequences, and can be used in the method of the invention as either anextrachromosomal element (e.g., a plasmid or vector) or as a stablechromosomal integrant. Exemplary modifying sequences include the C_(H)gene for use in isotype switching (i.e., replacement of the C_(H) geneof the target locus with a C_(H) gene of a different isotype orsubtype).

The precise mechanism through which intrachromosomal S-mediatedrecombination (also termed S--S recombination) occurs in theclass-switch phenomenon is not fully understood (for a review on thistopic, see Coffman et al., 1993, Adv. Immunol. 54:229-71). Without beingheld to a specific theory, naturally-occurring S-mediated recombinationis triggered by simultaneous transcription of two intrachromosomalswitch regions (Xu & Stavnezer (1990) Develop. Immunol. 1:11-17; Rothmanet al. (1990) Mol. Cell Biol. 10:1672-1679; Jung et al. (1993) Science159:984-987). For example, in a cell producing IgM antibody, the IgMheavy chain gene (which includes a VHDWJH region, a switch region (Sμ),and a Cμ gene) is constitutively transcribed and translated.Class-switching (e.g., to production of IgG) occurs when a second switchregion (e.g., Sγ) is transcribed. Transcription of a second switchregion is thought to be regulated by control elements associated witheach of the switch regions of the C_(H) locus. Each of these controlelements are activated by a different combination of cellular signals(i.e., one or more cellular signals) normally associated with cytokineswhich can be activated, for example, in a microbial infection orinflammation (e.g., cytokines such as interleukins, interferons, andtumor necrosis factor). In turn, production of cellular signals isassociated with specific types of infections and inflammation. Thus, aspecific type of infection or inflammation results in: 1) production ofa specific combination of cellular signals, which in turn determines 2)which of the switch region control elements is activated and, as aresult, 3) which switch region is transcribed to promote recombinationof its associated C_(H) region with the constitutively transcribed Sμand Cμ regions to produce a different, specific antibody isotype(Coffman et al., 1993, supra).

The present invention uses switch regions to provide a method ofdirecting recombination to pre-selected sites of interest in a mannerthat is not controlled by the normal cellular regulatory mechanismsdescribed above. As illustrated in FIGS. 4A-4C, the directed S--Srecombination of the present invention uses a targeting constructminimally containing a switch region (S₁) and a promoter (P₁), and atarget locus containing a switch region (S₂) and target sequence (T)under control of a promoter (P₂), to facilitate switch-site specificrecombination mediated by the two transcriptionally activated switchregions. The resulting recombinatorial product will minimally contain aswitch region having sequences from one or both switch regions, e.g.,S₁, or S₁ /S₂. When the targeting construct contains a promoter P₁ andS₁, the desired recombinatorial product will consist of the P₁ promoter,the switch region, and the target sequence, now under control of P₁instead of P₂ (FIG. 4A). The desired recombinatorial product isrecognized in a number of ways known to the art including PCR. When P₁is an inducible promoter, a cell containing the desired recombinatorialproduct can be recognized by induction of transcription. When thetargeting construct consists of P₁, S₁, and a modifying sequence, thedesired recombinatorial product will consist of the P₂ promoter, theswitch region, and the modifying sequence which replaces the targetsequence (FIG. 4B). The switch region may contain sequences from one orboth switch regions, e.g., S₁ or S₁ /S₂. When the modifying sequenceencodes a protein or peptide, the desired recombinatorial product can berecognized by synthesis of the desired product. When the targetingconstruct consists of P₁, DNA sequences 5' to S₁, and S₁, the desiredrecombinatorial product contains P₁ and the DNA sequences 5' to the S₁region inserted into the target locus (FIG. 4C). The desiredrecombinatorial product can be identified in a variety of ways,including PCR detection of the presence of the 5' DNA sequences and/orP₁, or by immunodetection technologies.

Additionally, the targeting construct can be used to insert a piece ofDNA 3' to a target locus contained in a specific chromosome. In thisembodiment, the targeting construct carries homologous sequencesallowing insertion into the selected chromosome by homologousrecombination. The resulting modified chromosome contains a DNA of thetargeting construct at a site 3' from the target locus. This embodimentis useful for induction of intrachromosomal S-mediated recombination.

Switch Regions

Class-switching (or isotype switching) results when B lymphocytesinitially expressing IgM switch their heavy chain isotype to IgG, IgA,or IgE upon maturation. Isotype switching results from a deletional DNArecombination event in which the C.sub.μ constant region of the heavychain, initially located downstream of the V_(H) D_(H) J_(H) region, isreplaced by a C.sub.γ, C.sub.α, or C.sub.ε constant regions (Rabbitts etal. (1980) Nature 283:351; Davis et al. (1980) supra; Kataoka et al.(1981) supra.

Several switch regions have been characterized, including the murine Sμ,Sε, Sα, Sγ₃, Sγ₁, Sγ_(2b) and Sγ_(2a) switch regions and the human Sμswitch region, such as Sμ₁ and Sγ₄ (Mills et al. (1995) J. Immunol.155:3021-3036, herein specifically incorporated by reference). Themurine Sμ region is about 3 kb and can be divided into a 3' region withsequences of [(GAGCT)nGGGGT]m, where n=1-7 and m=150 (Nikaido et al.(1981) supra), and a 5' region in which these two pentamers areinterspersed with the pentamer sequence (C/T)AGGTTG (Marcu et al. (1982)supra). The human Sμ locus is slightly different in that the heptamersequence is distributed throughout the region (Takahashi et al. (1982)supra; Mills et al. (1990) supra). Although other switch regions containmore complex patterns of repeated sequence, all switch sequences containmultiple copies of the pentameric sequences GAGCT and GGGGT (Nikaido etal. (1982) supra; Stanton et al. (1982) supra). The pentamers ACCAG,GCAGC, and TGAGC are also commonly found in switch regions (Gritzmacher(1989) supra). In addition, the heptameric repeat (C/T)AGGTTG isabundantly present in switch region sequences and is found near many,but not all, switch recombination sites that have been characterized inplasmacytomas and hybridomas (Marcu et al. (1982) supra).

The murine Sε and Sα loci contain 40 bp and 80 bp sequences,respectively, that are tandemly repeated. These sequences are homologousto Sμ, especially in areas of the repeats containing the GAGCT pentamer.Both human and murine Sγ regions are much less homologous to Sμ than arethe Sε and Sα regions. The homology of murine Sγ regions to Sμ decreaseswith the increasing distance 3' of the variable region(Sγ3>Sγ1>Sγ2b>Sγ2a). The murine Sγ regions are composed of tandemrepeats of 49 bp or 52 bp (Sγ2a), within which the pentameric sequencesTGGGG, GCAGC, and ACCAG are commonly found (Kataoka et al. (1981) supra;Mowatt et al. (1986) supra; Nikaido et al. (1982) supra, Nikaido et al.(1981) supra; Stanton et al (1982) supra; Szurek et al. (1985) supra; Wuet al. (1984) supra).

Switch regions suitable for use in the invention can be naturallyoccurring sequences, e.g., a switch region cloned directly from an Iglocus, preferably from a murine or human Ig locus. Alternatively, theswitch region can be a synthetically or recombinantly produced sequence.Recombinant switch regions can have the same sequence as a native,naturally-occurring switch region, or can be modified (e.g., containnucleotide substitutions, additions, mutations, and/or othermodifications) relative to a native switch region, with the proviso thatthe switch region retains its function in facilitating recombination.Recombinant switch regions can be designed to as to have a minimalnucleotide sequence necessary for switch-mediated recombination at thesame (or lower but acceptable) level as a native switch region, or at alevel enhanced relative to recombination promoted by a wild-type switchregion.

The switch-mediated recombination of the present invention providesimproved efficiency of S--S recombination over the naturally-occurringmechanism, as well as providing a widely application method of producinga desired protein. This is achieved, in part, with the use of promotersproviding constitutive or inducible transcription of the targetingconstruct, the target locus, or both the targeting construct and targetlocus. The improved efficiency of the switch-mediated recombinationmethod of the invention provides a frequency of recombination at a levelhigher than that which occurs naturally, that is, a 1% to 100% improvedefficiency; more preferably, a 20% to 100% improvement; and morepreferably a 50% to 100% improvement.

Targeting Constructs

As discussed above, targeting constructs of the invention are minimallycomposed of: 1) a switch region and 2) a promoter operably linked to and5' of the switch region. Additional optional components of the targetingconstruct include 3) a modifying sequence operably linked to and 3' ofthe switch region, including proteins, selectable markers, and/orcontrol elements, and/or 4) DNA sequences 3' to the promoter and 5' tothe switch region. Transcriptional activation of the promoter results inproduction of one or more translatable mRNA(s).

The Targeting Construct Promoter

The promoter of the targeting construct is selected according to thecell type in which directed S--S recombination is to be accomplished(e.g., a eukaryotic or prokaryotic cell, normally a eukaryotic cell).Because directed S--S recombination is dependent on transcription of theswitch regions of the targeting construct and the target locus, thepromoter of the targeting construct can be a constitutive or aninducible promoter. Suitable constitutive and strong constitutivepromoters for DNA expression in prokaryotic or eukaryotic cells are wellknown in the art. Where the cell in which directed S--S recombination isto take place is a eukaryotic cell, the promoter can be the heavy chainIg promoter or a viral promoter, such as a CMV, SV40, murine MoloneySarcoma virus (MMLV), and spleen-focus forming virus (SFFV) promoter, oran inducible promoter, such as MMTV and α-inhibin.

The Modifying Sequence

The modifying sequence can be any nucleic acid sequence that is suitablefor replacing a target sequence in a target locus. For example, themodifying sequence can be composed of a nucleotide sequence that encodesa translation product to replace all or a portion of the targetsequence. For example, where the target sequence is a C_(H) gene, themodifying sequence can be a different native C_(H) gene, a modifiedC_(H) gene (e.g, encoding an altered effector function relative to thewild-type C_(H) gene), or a native or modified light chain constantregion. Alternatively, the modifying sequence can encode anon-antibody-derived polypeptide that confers a function upon thepolypeptide encoded by the modified target sequence. For example, themodifying sequence can encode a toxin, hormone, growth factor, orportions thereof. The modifying sequence can also encode a linker toprovide covalent or non-covalent linkages between other (e.g., similarlymodified) heavy chain gene products or non-antibody polypeptides (e.g.,toxins, growth factors, hormones, or other biologically importantpolypeptide or other molecule). Yet another example of a modifyingsequence is a nucleotide sequence encoding a detectable polypeptidelabel or tag, e.g., β-galactosidase, alkaline phosphatase, horseradishperoxidase, or an immunodetectable polypeptide to which an antibody canbind to facilitate polypeptide detection and/or isolation (e.g., byimmunoaffinity chromatography).

Alternatively or in addition, the modifying sequence can containregulatory sequences (e.g., a promoter, enhancer element, an intron, ora ribosome binding site) that can be used to either introduce regulatorysequences at a position 3' of a switch region, or to replace regulatorysequences already present in the target sequence. For example,switch-mediated recombination can be used to replace a weak promoterwith a strong promoter in a target locus, where the weak promoter ispositioned 3' or 5' of the target locus switch region. Exemplaryregulatory sequences of particular interest in the modification of an Iglocus include a heavy chain enhancer sequence, a kappa chain enhancersequence, or a promoter derived from MMLV, Rous sarcoma virus (RSV), orSFFV.

The targeting construct may also contain an amplification gene thatallows the modified target locus to be amplified switch-mediatedproduct. There are a number of suitable amplification genes known to theart and useful in the invention, for example, the gene encodingdihydrofolate reductase (DHFR).

The modifying sequence is selected according to a variety of factorsincluding the target sequence to be modified, and/or the diagnostic ortherapeutic use intended for the resultant recombinatorial product.

Additional Sequences Present 3' of the Promoter and 5' of the SwitchRegion

The targeting construct can contain an additional, transcribable andtranslatable DNA sequence operably positioned between the promoter andswitch region of the target locus. This additional sequence can encodean N-terminal portion of the polypeptide encoded by the target locus.For example, the targeting construct can encode a desired V_(H) D_(H)J_(H) polypeptide. Upon directed switch-recombination with a targetlocus encoding an Ig heavy chain locus having a desired C_(H) gene atthe target sequence, the recombinatorial product contains the desiredV_(H) D_(H) J_(H) region and the desired C_(H) coding region, with theswitch region positioned between.

Other Components

The targeting construct can be based upon any of a variety of vectorsthat are well known in the art and commercially available (e.g., pBR322,pACYC vectors, plasmids, and viral vectors). "Vectors" include any DNAor RNA molecule (self-replicating or not) that can be used to transformor transfect a desired cell. The targeting construct can include othercomponents such as a selectable marker to facilitate screening andselection of cells containing the targeting construct as anextrachromosomal or chromosonally integrated element, and/or to selectfor cells that have successfully undergone directed S--S recombination,e.g., a selectable marker associated with the modifying sequence that isrecombined into the target locus in addition to the modifying sequence.Suitable selectable marker genes include genes encoding a detectablemarker (e.g., β-galactosidase) or drug resistance genes, e.g.,hygromycin resistance (hyg), guanosine phosphoryl transferase (gpt),neomycin resistance (neo), dihydrofolate reductase (DHFR), puromycin(spt) and ampicillin resistance (Amp). The construct can also include anorigin of replication for stable replication of the construct in abacterial cell (preferably, a high copy number origin of replication), anuclear localization signal, or other elements which facilitateproduction of the DNA construct, the protein encoded thereby, or both.For eukaryotic expression, the construct may also an amplification gene,which can increase levels of expression of the DNA of interest,particularly where the DNA of interest is a cDNA (e.g., contains nointrons of the naturally-occurring sequence). Any of a variety ofamplification genes known in the art may be used, including DHFR.

Target for Use with Targeting Constructs

As discussed above, a target locus suitable for use in the method of theinvention is minimally composed of: 1) a switch region, 2) a target DNAsequence adjacent and 3' of the switch region, and 3) a promoteroperably positioned in the construct to provide transcription of theswitch region and target sequence as an mRNA molecule. The target locuscan further contain an additional DNA sequence positioned adjacent and5' of the switch region; in such constructs, the promoter providestranscription of the additional DNA sequence, the switch region, and thetarget sequence as a one or more translatable mRNA(s).

In general, target loci suitable for use with the targeting constructsof the invention can be any switch-containing sequence in which theswitch region is transcribed and can facilitate switch-mediatedrecombination. The target locus can be any native, endogenouschromosomal sequence which contains a switch region (e.g., an Ig heavychain locus). Alternatively, the target locus can be an artificially,recombinantly produced sequence present as either an extrachromosomalelement (e.g., a vector or plasmid) or a chromosomally integratedelement. In a specific embodiment of the invention, where it isdesirable to insert a portion of a targeting construct 3' of a targetlocus on the same chromosome, the targeting construct carries homologoussequences directing recombination at a site 3' of a target locus.S-mediated recombination will then take place intrachromosomally, thusallowing controlled induction of intrachromosomal recombination.

The Promoter

The promoter of the target locus (P₂) can be the promoter that ispresent in the native, naturally-occurring target locus sequence and/orthe target sequence, or a promoter that is heterologous to the targetlocus sequence and/or the target sequence. Because S--S recombination isassociated with transcription of the switch region, the target locuspromoter preferably provides at least low-level expression, morepreferably constitutive expression, and even more preferably, provideshigh levels of constitutive expression of the target locus, specificallyof the switch region-encoding DNA. Where the promoter associated withthe target locus provides inadequate levels or undesirably low levels oftranscription of the switch region, the native target locus promoter canbe modified or replaced with a different promoter using S-mediatedrecombination or other recombinant methods well known in the art, e.g.,cloning, homologous recombination).

Additional Sequences Present 3' of the Promoter and 5' of the SwitchRegion

As discussed above, the target locus can contain an additional,transcribable and translatable DNA sequence operably positioned betweenthe promoter and switch region of the target locus. For example, theadditional sequences may encode an N-terminal portion of the polypeptideand the target locus contains a target sequence encoding the C-terminalportion of a polypeptide. After directed switch-mediated recombination,the modified target locus will contain both the N- and C-terminalportions of the polypeptide.

Other Components

The target locus can include additional components to facilitatereplication in prokaryotic and/or eukaryotic cells, integration of theconstruct into a eukaryotic chromosome, and markers to aid in selectionof and/or screening for cells containing the construct (e.g., thedetectable markers and drug resistance genes discussed above for thetargeting construct). For eukaryotic expression, the construct shouldpreferably additionally contain a polyadenylation sequence positioned 3'of the gene to be expressed. The polyadenylation signal sequence may beselected from any of a variety of polyadenylation signal sequences knownin the art. Preferably, the polyadenylation signal sequence is the SV40early polyadenylation signal sequence. Expression of the target locuscan also be enhanced by inclusion of intronic sequences, as discussedabove for the targeting construct.

An exemplary recombinant target locus of the invention is composed of(from 5' to 3'): 1) a promoter, 2) a first multiple cloning site forinsertion of a DNA sequence 5' to the switch region, 3) a switch region,and 4) a second multiple cloning site for insertion of a target DNAsequence. For example, where the target locus is a recombinant Ig heavychain gene, a V_(H) D_(H) J_(H) DNA sequence is inserted 5' to theswitch region at the first multiple cloning site, and the targetsequence is a C_(H) region inserted into the second cloning site.

Cell Lines Suitable for Use with the Method of the Invention

Any mammalian cell line capable of expressing the target locus ofinterest is suitable for use in the present invention. For example,where the target locus is an Ig heavy chain gene, the cell line is anymammalian cell capable of expressing a functional antibody. Ofparticular interest is the use of the switch-mediated recombinationmethod of the invention to facilitate class-switching inantibody-producing cells or cells with antibody-producing potential(e.g., stem cells). For example, the cell line can be, e.g., a hybridomacell line expressing human antibodies, an embryonic stem cell (e.g., amurine embryonic stem cell), a hybridoma cell line produced from B cellsfrom a transgenic animal (e.g., a transgenic mouse), or any other cell(normally a mammalian cell) capable of expressing at least a functionalportion of a heavy chain Ig locus or at least a functional portion of alight chain Ig locus. One example of a cell line useful in the method ofthe invention is a hybridoma cell line expressing human antibodiesderived from B cells from the Xenomouse (Green et al. (1994) NatureGenetics 7:13 and PCT patent publication No. WO 94/02602, both of whichare herein specifically incorporated by reference). The Xenomousecarries large segments of the human heavy chain and K chain lociintegrated into its germline, as well as containing functionallyinactivated mouse heavy and kappa light chain alleles. Xenomouseproduces B cells expressing human heavy chain (hμ) and human K lightchain (mK), or hμ and mouse lambda (mλ) light chain. Co-expression of hκand mλ does not occur, since expression of one light chain completelyexcludes the expression of the other (Green et al. (1994) supra). Uponimmunization, Xenomouse produces a broad adult-like repertoire of humanIg and give rise to antigen-specific human monoclonal antibodies.Xenomouse allows generation of mouse hybridomas making antigen-specifichuman monoclonal antibodies. Methods for producing hybridoma cell linesare well known in the art (see, for example, Harlow and Lane, eds.,1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.). Methods for producing cell lines expressinghuman or "humanized" antibodies are also well known in the art (see, forexample, PCT Publication Nos. WO 94/02602 and WO 91/10741).

Where the cell line is an antibody-producing lymphoid cell line, thecell line can express the antibody from either a genomic sequence, amodified sequence, a heterologous sequence (e.g., an Ig sequence fromanother species), a modified heterologous sequence, or a chimericsequence (e.g., composed of both murine and human Ig sequences). Thus,the cell line can be, for example, a murine hybridoma cell lineproducing either a murine, human, or chimeric antibody. The hybridomacell line can be producing human antibodies by, for example, expressionof human Ig genes. In one embodiment, the cell is a murine lymphoid cellproducing a human antibody by expression of human Ig genes. In onevariation of the embodiment, the constant region gene of the genomicsequence is a human constant (hC_(H)) region gene, e.g., a hC_(H) geneof the mu class (hC_(H) μ), and the modifying sequence is a humanconstant region of the gamma class (hC_(H) λ).

Methods Using Switch-Mediated Recombination

Switch-mediated recombination using the construct(s) of the inventioncan be accomplished in a variety of ways. For example, 1) the targetlocus can be naturally occurring (chromosomally located) and thetargeting construct can be used as either an extrachromosomal orchromosomally integrated element; or 2) the target locus can be anaturally occurring or recombinantly produced sequence that is eitherpresent as an extrachromosomal or a chromosomally integrated element,and the targeting construct can be used as either an extrachromosomal orchromosomally integrated element. When the targeting construct andtarget locus are both chromosomally integrated, they are integrated onthe same or different chromosomes.

Switch-Mediated Recombination Using a Chromosomal Target Locus and aChromosomally Integrated Targeting Construct

In this embodiment, the cell line used to accomplish directedswitch-mediated recombination either: 1) contains an endogenous,naturally occurring target locus, or 2) contains a chromosomallyintegrated recombinant target locus. Methods for introduction of DNAinto a host cell and selection for stable chromosomal integrantscontaining a specific DNA sequence of interest are well known in the art(see, for example, Sambrook, et al.,1989, Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; hereby incorporated by reference with respect tomethods and compositions for recombinant DNA techniques to provide atransformed cell containing a stably integrated DNA of interest, andexpression of the DNA of interest).

The targeting construct can be linearized, e.g., by digestion with arestriction endonuclease(s), and the linear DNA introduced into the hostcell using any of a variety of methods known in the art (e.g.,electroporation, microinjection, liposome fusion, red blood cell ghostfusions, protoplast fusion, yeast cell fusion, or any other method knownin the art (see, for example, Sambrook et al., supra)). The linearvector is then integrated into the cell's genome randomly orspecifically by directed homologous recombination, and stable integrantsare selected by, for example, expression of a selectable markerassociated with the targeting construct, or by expression of themodifying sequence in the targeting construct.

Directed switch-mediated recombination is accomplished by simultaneoustranscription of the switch regions in the target locus and thetargeting vector. Cells containing the recombinatorial product, forexample, the modified target locus, are identified and selected byexpression of the modified target locus gene product (e.g., by ELISAreactivity or fluorescence-activated cell sorting (FACS)).

Switch-Mediated Recombination Using a Chromosomal Target Locus and anExtrachromosomal Targetina Construct

In this emboiment of the method of the invention, the targetingconstruct is introduced-into the cell containing a chromosomallyintegrated target locus by methods well known in the art (see, forexample, Sambrook et al., 1989, supra). In contrast to the methodimmediately above, the targeting construct is maintained as anextrachromosomal element for a time sufficient for transcription of thetargeting construct's switch region and recombination with thetranscriptionally active switch region of the target locus. Cellscontaining the desired recombinatorial product, e.g., a modified targetlocus, can be identified and selected as described above, e.g.,selection for expression of a selectable marker associated with theintegrated target sequence, or detection of cells expressing the desiredmodified target locus gene product.

Screening and Selection

Detection of properly recombined sequences can be accomplished in avariety of ways, depending upon the nature of the desiredrecombinatorial product. For example, where the modifying sequenceassociated with a selectable marker is recombined into the target locuswith the modifying sequence, an initial screen will select for thosecells which express the marker. A second screen can be used to determineif the drug resistant cells express the appropriately modified targetlocus.

The method used for the second screen will vary with the nature of themodifying sequence inserted into the target locus. The modifyingsequence can be detected by Southern blot using a portion of themodifying sequence as a probe, or by polymerase chain reaction (PCR)using amplifying primers derived from the modifying and modifiedregions. The cells having an appropriately integrated modifying sequencecan also be identified by detecting expression of a functional modifiedtarget locus product, e.g., immunodetection of the new C_(H) region in amodified antibody heavy chain locus. Alternatively, the expressionproduct of the modified target locus can be detected using a bioassay totest for a particular effector function conferred by the modifyingsequence. For example, the expression of modifying sequence that encodesa biologically active molecule such as an enzyme, toxin, growth factor,or other peptides is assayed for that particularly biological activity.

Where the target locus is an Ig gene, the product of the modified targetlocus can also be tested for appropriate antigen or ligand recognitionvia any conventional immunological screening methods known in the art,e.g, ELISA, FACS, antibody-dependent cell cytotoxicity assays, orimmunoprecipitation assays (see, for example, Harlow and Lane, supra).

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use various constructs and perform the various methods of thepresent invention and are not intended to limit the scope of what theinventors regard as their invention. Unless indicated otherwise, partsare parts by weight, temperature is in degrees centigrade, and pressureis at or near atmospheric pressure. Efforts have been made to ensureaccuracy with respect to numbers used, (e.g., length of DNA sequences,molecular weights, amounts, particular components, etc.) but somedeviations should be accounted for.

Example 1

Targeting Construct for Switch-Mediated Recombination (PTSW-1.4 andpTSW-1.9)

All the vectors generated were based on a low copy-number pACYC177Plasmid (NEB). The vector pTSW-1.4 was generated from the p1bYACδNotplasmid containing a 23 kb EcoRI genomic fragment of the entire human γ2switch region, isolated from human placenta genomic library. Thisfragment contained 2 kb of coding sequences, 12 kb of upstream sequencesincluding the I exon and γ2 switch region, and 9 kb of downstreamsequences (Flanagan & Rabbitts (1982) Nature 300:709-713). This plasmidalso contains the mouse 3' enhancer (Dariavach et al. (1991) Eur. J.Immunol. 21:1499-1504). The vector was modified to contain a hygromycinselectable marker and a human CMV promoter-enhancer cassette, whichincluded at its 3' end prokaryotic terminator sequences (describedbelow). The prokaryotic terminator sequences were used to stopfortuitous prokaryotic transcripts from activating the switch sequences,and thus destabilizing them during cloning in bacteria (Mowatt & Dunnick(1986) J. Immunology 136:2674-2683). These sequences were confirmed tohave very little effect on eukaryotic transcription.

The hygromycin gene, driven by the SV40 promoter (Giordano & McAllister(1990) Gene 88:285-288) was cloned as a 1.7 kb HindIII-BamHI fragmentfrom pUC219. TG76 plasmid and inserted into HindIII and BamHI sites inpACYC177 to generate pACYC.hyg plasmid.

The terminator was synthesized asGCATGCCCGCGG-GAATAGGCGGGCTTTTTTNNNGCCGCGGCTCGA (SEQ ID NO:1), withflanking SphI sites, and an internal XhoI site at the 3' end, forcloning purposes. This sequence was cloned into the SphI site ofpIK1.1Cat plasmid, downstream of the human CMV promoter-enhancersequences.

The CMV expression cassette, together with the terminator sequences, wascloned as a 900 bp HindIII-XhoI fragment, which was placed into theHindIII and XhoI sites in the pACYC.hyg plasmid described above togenerate pACYC.hyg.CMVt, in which the CMV transcription orientation isopposite to that of the hygromycin gene.

The 2.6 kb fragment, containing both hygromycin and CMV-terminatorcassettes, was excised from pACYC.hyg.CMVt by BamHI and XhoI digestion.Both ends of this fragment were converted into NotI sites, usinglinkers, and the fragment was cloned into the unique NotI site inp1bYACδNot plasmid containing 23 kb human γ2 sequences and mouse 3'enhancer. pTSW-1.4 plasmid (FIG. 5) was generated with CMV transcriptionorientation in the same direction as that of the human γ2 codingsequences. pTSW-1.9 plasmid (FIG. 6) was generated with CMVtranscription orientation opposite to that of the human γ2 codingsequences.

An exemplary targeting construct of the invention, designated pTSW-1.4,is shown in FIG. 5. pTSW-1.4 is constructed for use in switch-mediatedreplacement of an Ig heavy chain constant gene with a human heavy chainIgG₂ constant gene (hCHγ2). The pTSW-1.4 construct contains a CMVpromoter operably linked to, in the 5' to 3' orientation, the human IgG2heavy chain region (approximately 23 kb), which includes the IgG2 heavychain I exon and its 5' flanking sequences, the human IgG2 switchregion, the complete human hCHγ2 gene, and sequences flanking the IgG2heavy chain region. The hCHγ2 region is linked to a murine enhancerpositioned adjacent and 3' of the hCHγ2 gene. The CMV promoter is astrong constitutive promoter. Other constitutive promoters can be usedinstead of the CMV promoter (e.g., SSFV, MMLV, MCV, RSV, SV40, etc.).Both the hCHγ2 regions have been cloned and sequenced (Mills et al.(1995) supra). Murine 3' enhancer is also well known in the art(Dariavach et al. (1991) supra).

Example 2

Targeting Construct for Switch-Mediated Recombination (pTSW-2)

To generate pTSW-2 plasmid, a 13 kb BamHI fragment was cloned from the23 kb EcoRI human γ2 genomic fragment in p1bYACδNot plasmid, asdescribed in Example 1, followed by partial fill-in reaction with Klenowto generate fragment ends compatible with XhoI. This clone was insertedinto the unique XhoI site in pACYC.hyg.CMVt plasmid, which was alsopartially filled in with Klenow to make the site compatible with BamHI.The correct orientation of the clone, in which the transcriptionorientation of human γ2 coding sequences is the same as the CMVpromoter, was selected.

Another exemplary targeting construct of the invention, designatedpTSW-2, is shown in FIG. 7. Like pTSW-1.4, pTSW-2 is constructed for usein switch-mediated replacement of an Ig heavy chain constant gene with ahuman heavy chain IgG₂ constant gene (hCHγ2). The pTSW-2 construct isprepared using a CMV promoter operably linked to, in the 5' to 3'orientation, the human IgG2 heavy chain region (approximately 13 kb)including the human IgG2 switch region and 200 bp 5' flanking sequencesof the switch region and human γ2 open reading frame. Some of theflanking sequences present in pTSW-1.4 are not present in pTSW-2. ThepTSW-2 construct also contains the selectable marker SV2hyg and aprokaryotic transcription terminator (to stabilize the switch region).The pTSW-2 construct can be prepared either with or without a murineenhancer positioned 3' of the hCHγ2 gene.

Example 3

TargetinG Construct for Switch-Mediated Recombination (RTSW-3.1)

To generate pTSW-3.1 plasmid (FIG. 8), 2 kb of human γ2 coding sequenceswere cloned by PCR from p1bYACδNot as an XhoI-SalI fragment (Example 1).This fragment was cloned into the unique XhoI in pACYC.hyg.CMVt plasmid,3' of the terminator sequences. Mouse γ1 switch sequences were excisedas a 10 kb HindIII-EcoRI fragment from p-gamma-1/EH10.0 plasmid (Mowatt& Dunnick (1986) supra), and the ends were converted into XhoI and SalI,respectively. The modified plasmid was cloned 5' of the human γ2 regionvia the unique XhoI site in pACYC.hyg.CMVt. pTSW-3.1dBglII plasmid (FIG.9) was generated similarly to pTSW-3.1, except that a 7.9 kb BglII-EcoRImouse γ1 switch sequences was included.

pTSW-3.2 was constructed as described for pTSW-3.1, except that the CMVpromoter-enhancer cassette was replaced by the spleen focus formingvirus (SSFV) promoter.

pTSW-3 plasmids contained unique NotI and MluI sites (converted by alinker from the unique BamHI site). HindIII is used for linearizationand for cloning of the 3' enhancer.

A further exemplary targeting construct of the invention, designatedpTSW-3.1, is shown in FIG. 8. Like pTSW-1.4 and TSW-2, pTSW-3.1 isconstructed for use in switch-mediated replacement of an Ig heavy chainconstant gene with a human heavy chain IgG₂ constant gene (hCH.sub.γ2).The pTSW-3.1 construct is prepared using a CMV promoter operably linkedto, in the 5' to 3' orientation, a murine γ1 switch region which maycontain also the mouse γ1 I exon and flanking sequences, and a humangenomic constant hCH.sub.γ1, hCH.sub.γ2, or hCH.sub.γ4 coding sequence,which includes the 5' flanking branch point and splice acceptor. ThepTSW-3.1 construct can optionally further contain a murine γ1 controlelement (mI.sub.γ1) positioned adjacent and 5' of the mS.sub.γ1,sequence. Alternatively, the human switch region of the γ1 gene(hS.sub.γ1) and its 5' flanking sequences, such as the I exon(hI.sub.γ1) can be used instead of the mI.sub.γ1, and mS₁. In constructswhich do not contain I exon, a splice donor site is provided 3' of thepromoter sequences. The pTSW-3.1 construct may further optionallycontain a murine 3' enhancer positioned adjacent and downstream of thehC_(H)γ gene and/or a 3' eukaryotic transcription terminator positioned3' and adjacent the hC_(H)γ gene. Each of the elements of the pTSW-3construct are well known in the art (mouse S.sub.γ4, S.sub.μ, Mills etal. (1991) supra; mouse S.sub.γ1, Mowatt & Dunnick (1986) supra; humanSγ, Mills et al. (1995) supra; mouse 3' enhancer, Dariavach et al.(1991) suDra). The pTSW-3.1 construct also contains the selectablemarker SV2γ2hyg and a HindIII linearization site. Other selectablemarkers may be used, for example, pyromycin.

Example 4

Switch-Mediated Recombination in a Hybridoma Cell Line

As discussed above, one of the problems associated with production ofhuman monoclonal antibodies is that the immortal cell line fused withthe human B cells is of murine origin. This can result in arecombinatorial event where the resulting antibody has a human variableregion (human light chains and human heavy chain variable region (hV_(H)D_(H) J_(H))), but has a murine heavy chain constant region (mC_(H)γ)(see FIG. 8). Switch-mediated recombination is used to replace themC_(H) γ gene with a human heavy chain constant region (hC_(H)γ) gene.

A hybridoma cell line expressing a monoclonal IgG antibody againstantigens, including human antigens, having a human heavy chain variableregion (hV_(H) D_(H) J_(H)) and a murine heavy chain constant region(mC_(H)γ) is produced using methods well known in the art (for example,see, Green et al. (1994) supra). A targeting construct containing apromoter operably linked to a switch region and the hC_(H)γ gene isconstructed as described above. Any of the exemplary vectors describedin the examples above (pTSW-1.4, pTSW-2, or pTSW-3.1) is suitable foruse in this method. The construct is linearized, and the linearconstruct introduced into the hybridoma cell by, for example,electroporation, lipofection, or other methods known to the art. Thetransfected hybridoma cells, containing stable integrants of theconstruct, are selected by their ability to grown in hygromycin.Hygromycin resistant cells are then cultured further to allow fortranscription of the target construct from the CMV promoter and theresulting switch-mediated recombinatorial event. Hybridoma single cellcultures are then screened for expression of hC_(H)γ2 by amplificationof recombined antibody message or by using an anti-human IgG2 antibodyin a sandwich ELISA assay, or isolated by FACS sorting.

Example 5

Switch-Mediated Recombination in a Transgenic Mouse Producing HumanAntibodies

Switch-mediated recombination may be accomplished in a transgenic mousein vivo as follows. The targeting vector is introduced as a transgeneinto a human antibody-producing mouse and the recombined antibodies,produced by mouse B cells or their derived hybridomas, are screened asdescribed.

The instant invention is shown and described herein in what isconsidered to be the most practical, and preferred embodiments. It isrecognized, however, that departures may be made therefrom which arewithin the scope of the invention, and that modifications will occur toone skilled in the art upon reading this disclosure.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 1                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GCATGCCCGC GGGAATAGGC GGGCTTTTTT NNNGCCGCGG CTCGA   - #                      - #45                                                                    __________________________________________________________________________

What is claimed is:
 1. An isolated cell comprising an artificial nucleicacid cargeting construct, the targeting construct comprising:a switchregion; a promoter operably linked to and 5' of the switch region; and amodifying sequence operably linked to and 3' of the switch region;wherein the cell facilitates switch-mediated recombination when theswitch region of the targeting construct and a switch region of a targetlocus are transcribed.
 2. The cell of claim 1, wherein the modifyingsequence encodes a constant region of an antibody heavy chain.
 3. Thecell of claim 1, wherein the modifying sequence encodes a constantregion of a murine antibody heavy chain.
 4. The cell of claim 1, whereinthe modifying sequence encodes an immunoglobulin variable region, apromoter, an enzyme, a toxin, a hormone, a growth factor, a detectablepeptide label, or a linker polypeptide.
 5. The cell of claim 1, whereinthe cell is an antibody-producing cell or a cell with antibody-producingpotential.
 6. The cell of claim 1, wherein the cell is selected from thegroup consisting of an embryonic stem cell and a lymphoid cell.
 7. Thecell of claim 1, wherein the cell is a bacterial cell.
 8. The cell ofclaim 2, wherein the constant region is the constant region of a humanantibody heavy chain.
 9. The cell of claim 2, wherein the constantregion is selected from the group consisting of Cγ, Cμ, Cα, Cδ, and Cε.10. A method for performing directed switch-mediated recombination, themethod comprising the steps of:introducing a targeting construct into anisolated B cell or B cell hybridoma,wherein the targeting constructcomprises a first switch region, and a promoter (P₁) operably linked toand 5' of the first switch region, and wherein the cell comprises atarget locus comprising a second switch region, a target sequenceadjacent and 3' of the second switch region, and a promoter (P₂)operably positioned with in the target locus to provide transcription ofthe second switch region and target sequence; and culturing the cell toallow transcription of the first and second switch regions, therebypromoting recombination between the first switch region of the targetingconstruct and the second switch region of the target locus; andselecting a cell comprising a modified target locus comprising P₁, aswitch region, and the target sequence; wherein P₁, the switch region,and the target sequence are operably linked and wherein the targetsequence is under control of P₁, indicating that directedswitch-mediated recombination has occurred.
 11. An isolated B cell or Bcell-derived hybridoma produced by the method of claim
 10. 12. Themethod of claim 10, wherein the targeting construct and the target locusare chromosomally integrated.
 13. The method of claim 10, wherein thetarget locus encodes an antibody heavy chain.
 14. The method of claim10, wherein the target locus further comprises a non-target sequencepositioned between the target locus promoter and the target locus switchregion.
 15. The method of claim 10, wherein the targeting construct islinearized prior to said introducing, and a cell containing a stableintegrant of the targeting construct is selected prior to said culturingto allow target locus transcription.
 16. The method of claim 10, whereinthe targeting construct is extrachromosomal.
 17. The method of claim 12,wherein recombination between the first switch region and the secondswitch region is associated with deletion of nucleic acid locatedbetween said first and second switch regions.
 18. The method of claim14, wherein the non-target sequence encodes a human antibody heavy chainvariable region, and the target sequence encodes a murine antibody heavychain constant region.
 19. A method for performing directedswitch-mediated recombination, the method comprising the stepsof,introducing a targeting construct into an isolated B cell or B celhybridoma that facilitates switch-mediated recombination when two switchregions are transcribed, where in the targeting construct comprises afirst switch region, and a promoter (P₁) operably linked to and 5' ofthe first switch region, and a modifying sequence operably linked to and3' of the first switch region, wherein the cell comprises a target locuscomprising a second switch region, a target sequence adjacent and 3' ofthe second switch region, and a promoter (P₂) operably positioned withinthe target locus to provide transcription of the second switch regionand target sequence; and culturing the cell to allow transcription ofthe first and second switch sequences, thereby promoting recombinationbetween the first switch region of the targeting construct and thesecond switch region of the target locus; and selecting a cellcomprising a modified target locus comprising P₂, a switch region, andthe modifying sequence; wherein P₂, the switch region, and the modifyingsequence are operably linked, indicating that directed switch-mediatedrecombination has occurred.
 20. The method of claim 19, wherein thetarget locus encodes an antibody heavy chain.
 21. The method of claim19, wherein the target locus encodes a murine antibody heavy chain andthe modifying sequence of the targeting construct encodes a humanantibody heavy chain constant region.
 22. The method of claim 19,wherein the target locus further comprises a non-target sequencepositioned between the target locus promoter and the target locus switchregion.
 23. The method of claim 19, wherein the targeting construct islinearized prior to said introducing, and a cell containing a stableintegrant of the targeting construct is selected prior to said culturingto allow target locus transcription.
 24. The method of claim 19, whereinthe targeting construct is extrachromosomal.
 25. An isolated B cell or Bcell-derived hybridoma produced by the method of claim
 19. 26. Themethod of claim 19, wherein the targeting construct and the target locusare chromosomally integrated.
 27. The method of claim 22, wherein thenon-target sequence encodes a human antibody heavy chain variableregion, the target sequence encodes a murine antibody heavy chainconstant region, and the modifying sequence of the targeting constructencodes a human antibody heavy chain constant region.
 28. The method ofclaim 26, wherein recombination between the first switch region and thesecond switch region is associated with deletion of nucleic acid locatedbetween said first and second switch regions.
 29. A method for producinga modified antibody heavy chain by switch-mediated recombination, themethod comprising the steps of:a) introducing a targeting construct intoan isolated B cell or B cell hybridoma that facilitates switch-mediatedrecombination, wherein the targeting construct comprises a switch region(S₁), a promoter operably linked to and 5' of S₁, and a modifyingsequence operably linked to and 3' of S₁, andwherein an antibody heavychain expressed by the cell is encoded by an antibody heavy chain locuscomprising a promoter, an antibody heavy chain variable region operablylinked to and 3' of the promoter, a switch region (S₂) adjacent and 3'of the variable region, and an antibody heavy chain constant regionadjacent and 3' of S₂ ; b) culturing the cell to allow transcription ofthe targeting construct, wherein switch-mediated recombination betweensaid S₁ and S₂ is promoted; and c) selecting a cell comprising amodified heavy chain locus, the modified heavy chain locus comprisingthe heavy chain locus promoter, the heavy chain variable region, aswitch region, and the modifying sequence of the targeting construct,wherein the heavy chain locus promoter, the variable region, the switchregion, and the modifying sequence are operably linked;wherein amodified antibody heavy chain is produced.
 30. An isolated B cell or Bcell-derived hybridoma produced by the method of claim
 29. 31. Anisolated B cell or B cell-derived hybridoma comprising a genomecomprising a switch-mediated recombinatorial product, the product havingbeen produced via switch-mediated recombination between an exogenousnucleic acid targeting construct and a target locus.